Cerebroside Sulfatase Research Articles

Steroid sulfatase, arylsulfatases A and B, galactose-6-sulfatase, and iduronate sulfatase in mammary cells and effects of sulfated and non-sulfated estrogens on sulfatase activity

Sulfatase enzymes have important roles in metabolism of steroid hormones and of glycosaminoglycans (GAGs). The activity of five sulfatase enzymes, including steroid sulfatase (STS; arylsulfatase C), arylsulfatase A (ASA; cerebroside sulfatase), arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase), galactose-6-sulfatase (GALNS), and iduronate-2-sulfatase (IDS), was compared in six different mammary cell lines, including the malignant mammary cell lines MCF7, T47D, and HCC1937, the MCF10A cell line which is associated with fibrocystic disease, and in primary epithelial and myoepithelial cell lines established from reduction mammoplasty. The effects of estrogen hormones, including estrone, estradiol, estrone 3-sulfate, and estradiol sulfate on activity of these sulfatases were determined. The malignant cell lines MCF7 and T47D had markedly less activity of STS, ASB, ASA, and GAL6S, but not IDS. The primary myoepithelial cells had highest activity of STS and ASB, and the normal epithelial cells had highest activity of GALNS and ASA. Greater declines in sulfatase activity occurred in response to estrone and estradiol than sulfated estrogens. The study findings demonstrated marked variation in sulfatase activity and in effects of exogenous estrogens on sulfatase activity among the different mammary cell types.

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.

Degenerative Disorders of the Central Nervous System

Degenerative Disorders of the Central Nervous System

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.

Arylsulfatases A and B in EBV-transformed lymphoid cell lines: Studies on their molecular forms in cells from patients with inborn sulfatase deficiencies. Comparative diagnostic value of enzymatic assays

The enzyme activity of arylsulfatase A and arylsulfatase B was studied in Epstein-Barr virus-transformed lymphoid cell lines established from control individuals and patients affected with metachromatic leukodystrophy, mucopolysaccharidosis type VI (or Maroteaux-Lamy syndrome) and multiple sulfatase deficiency. Lymphoid cells derived from patients with metachromatic leukodystrophy showed a severe deficiency in cerebroside sulfatase activity, as measured using radiolabelled sulfatide, but some residual activity of arylsulfatase A when measured with the chromogenic substrate, para-nitrocatechol sulfate. Lymphoid cells from mucopolysaccharidosis type VI had virtually no arylsulfatase B activity. In cells from patients with multiple sulfatase deficiency, the activities of lysosomal sulfatases as well as steroid sulfatase were deficient. Study of the molecular forms of arylsulfatases confirmed the complete deficiency of arylsulfatase A and arylsulfatase B activities in metachromatic leukodystrophy and mucopolysaccharidosis type VI lymphoid cells, respectively. The arylsulfatase A defect in metachromatic leukodyslymphoid cells, respectively. The arylsulfatase A defect in metachromatic leukodystrophy cells could be demonstrated on focused fractions even using the artificial substrates, para-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. To investigate the discrepancy of the arylsulfatase A activity data observed between whole cell homogenates and focused fractions when using the synthetic substrates, assays were tentatively performed for optimizing the determination of arylsulfatase A on crude homogenates of lymphoid cells. Although this work has indicated methodological limitations of the enzymatic assay of arylsulfatase A in lymphoid cells using methylumbelliferyl sulfate, it emphasizes the validity of lymphoid cell lines as an experimental model for the study of inborn deficiencies of arylsulfatases A and B.

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.

Activator-Dependent Hydrolysis of Myelin Cerebroside Sulfate by Arylsulfatase A

A purified myelin preparation containing [35S]-labeled cerebroside sulfate (CS) was biosynthesized in developing rat brain and tested as a model of a physiological substrate for CS hydrolysis by arylsulfatase A. Particular attention was directed to the involvement of the CS sulfatase activator protein in facilitating the catabolic process. Although arylsulfatase A alone was incapable of desulfating CS in either purified CS suspensions or the physiological membrane, activator-induced hydrolysis of myelin CS exhibited concentration dependency, pH optimum, and relative insensitivity to salts in a manner similar to purified lipid suspensions. Exogenous protein demonstrated concentration-dependent inhibition. Slower rates of hydrolysis observed for the myelin membrane substrate are proposed to be a consequence of myelin membrane configuration and competition for activator by other lipoidal constituents.

Galactosylceramide sulfotransferase, arylsulfatase A and cerebroside sulfatase activity in different regions of developing rat brain

The in vivo metabolism of sulfatides was studied in spinal cord and cerebral cortex of developing rat pups. Developmental changes in the rate of sulfolipid synthesis were measured after the intraperitoneal injection of 35SO 4 2−. We also measured the accumulation of sulfatides, as well as the profiles of cerebroside sulfotransferase, cerebroside sulfatase and arylsulfatase A in both brain regions as a function of postnatal development. The accumulation of sulfatides was higher in spinal cord than in cerebral cortex. In addition, sulfatide metabolism was more active in spinal cord. In both brain regions, the developmental pattern of 35SO 4 2− incorporation into sulfolipids was closely correlated to the activities of cerebroside sulfotransferase and of arylsulfatase A. The activity of these enzymes was initially low, increased during the period of active myelination and declined thereafter. However, the activity of cerebroside sulfatase, measured with its physiological substrate, [ 35S]sulfatide, increased during development and did not decline. An explanation for the difference between the developmental profiles of the arylsulfatase A and cerebroside sulfatase reactions (which are supposed to be catalysed by the same enzyme) is proposed.

Comparison of the Mechanisms of Action of Insulin and Triiodothyronine on the Synthesis of Cerebroside Sulfotransferase in Cultures of Cells Dissociated from Brains of Embryonic Mice

The effect of low (physiological) concentrations of insulin (2 and 20 ng/ml) and L-triiodothyronine (T3) were studied on two myelin-related enzymes: (1) the 3'-phosphoadenosine-5'-phosphosulfate:cerebroside sulfotransferase (CST, EC 2.8.2.11) catalyzing the production of sulfatide, and (2) the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP, EC 3.1.4.3.7) in myelinogenic cultures of cells dissociated from embryonic mouse brain. Insulin treatment (20 ng/ml) of the cells in the presence of serum increased CST activity at 18 and 25 days in vitro (DIV) by 86 and 211%, respectively. At 18 DIV and under the same conditions, CNP was significantly stimulated (95%) by high doses of insulin (2,000 ng/ml) only, while arylsulfatase A (EC 3.1.6.1) or cerebroside sulfatase activities, both of which are involved in sulfatide degradation, were unchanged. Thus, it can be assumed that the observed increase of the incorporation of [35S]O4 into sulfatide after insulin treatment of mixed cell cultures is the result of CST induction rather than a decreased catabolism. The level of CST activity in insulin-treated cells (20 ng/ml) in serum-free medium was also increased at 18 and 25 DIV by about 50 and 70%, respectively. Conversely, none of the insulin concentrations used in the absence of serum (even at high doses) had any effect, either at 18 or 25 DIV on CNP and ASA activities. The involvement of insulin in the regulation of sulfatide synthesis was further confirmed by dose-response curves relating the activity of CST to hormone concentration in the medium. The increase in the activity of CST in insulin-treated cells was due only to the increase in the Vmax of this enzyme, suggesting that it may be attributed to enzyme induction. A study of kinetic parameters of CST indicated that there were no differences in pH optimum and Km values between control and induced enzyme. Further experiments using cycloheximide point to a direct effect of insulin on oligodendrocyte CST induction. Data similar to those described above for insulin were also obtained with T3. As for insulin, T3 stimulated the induction of CST but in serum-free medium only. This effect was prevented by cycloheximide. In addition, the induction of CST by T3 was blocked by actinomycin D. This was not the case for insulin. These results suggest that T3 and insulin act on CST by different mechanisms, i.e. at transcriptional and post-translational levels, respectively. Apart from this, the insulin effect on CST activity was additive to that of T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. Deficiency in urine of patients affected with cerebroside sulfatase activator deficiency and identity with activators for the enzymatic hydrolysis of GM1 ganglioside and globotriaosylceramide.

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.

Genetic complementation in somatic cell hybrids of cerebroside sulfatase activator deficiency and metachromatic leukodystrophy fibroblasts.

Several cases of metachromatic leukodystrophy (MLD) have been described with normal or near normal activities of arylsulfatase A (cerebroside sulfatase). However, the ability of intact cultured fibroblasts to hydrolyze cerebroside sulfate was impaired. Since the impairment was corrected by cerebroside sulfatase activator, a deficiency of activator was implied. In the absence of direct demonstration of deficiency, other types of evidence were needed to support the premise that the genetic defect was not associated with the arylsulfatase A locus as in classical MLD. Therefore, somatic cell hybrids of activator deficiency and MLD fibroblasts were analyzed. Complementation was indicated by enhanced hydrolysis of cerebroside sulfate, supporting the view that cerebroside sulfatase activator deficiency and MLD are nonallelic.

Microheterogeneity of arylsulfatase A: Treatment with hydrolytic enzymes

Cerebroside sulfatase also known as arylsulfatase A from human liver displays six microheteromer bands upon narrow pH range isoelectric focusing. Sialic acid residues only partially account for this enzyme multiplicity since neuraminidase treatment reduces the number of bands to three. Uptake studies with cultured fibroblasts strongly suggest arylsulfatase A has covalently bound mannose 6-phosphate residues. However, treatment with alkaline phosphatase and a battery of glycohydrolases failed to reduce the number of enzyme charge forms below three. These results imply that the neuraminidase-resistant charge microheterogeneity is not due to structures associated with the carbohydrate moiety of arylsulfatase A.

Juvenile and adult metachromatic leukodystrophy: partial restoration of arylsulfatase A (cerebroside sulfatase) activity by inhibitors of thiol proteinases.

Arylsulfatase A polypeptides were examined in cultured fibroblasts from a patient with juvenile metachromatic leukodystrophy and three patients with the adult form of the disease, with the aid of metabolic labeling and immunoprecipitation. The mutant cells were severely deficient in the arylsulfatase polypeptides. The apparent rate of synthesis, however, as estimated from the secretion of polypeptides or activity by cells incubated in the presence of 10 mM NH4Cl was 20-50% of control. In the absence of NH4Cl, the mutant enzyme was rapidly degraded upon transport into lysosomes. In the presence of inhibitors of thiol proteinases arylsulfatase A polypeptides were partially protected from degradation, and the catalytic activity of arylsulfatase A was increased. In addition, the treatment partially corrected the capacity of the cells to degrade cerebroside sulfates. Inhibitors of thiol proteinases may be of therapeutic value in variants of metachromatic leukodystrophy, in which an unstable arylsulfatase A is synthesized.

Impaired cerebroside sulfate hydrolysis in fibroblasts of sibs with "pseudo" arylsulfatase A deficiency without metachromatic leukodystrophy.

Low arylsulfatase A levels are reported in two siblings, one with a neurologic disability not typical for metachromatic leukodystrophy, the other a healthy 18-year-old female with a normal developmental history. In both individuals, arylsulfatase A levels in white blood cells were 7-8% of control values. Cultured fibroblasts gave low values (8-10% of normal) for both cerebroside sulfatase and arylsulfatase A activities. Other family members had enzyme levels consistent with heterozygote or normal status. Cerebroside sulfate loading tests of cultured fibroblasts in 199-CO2 media were normal for all family members who were tested. In MEM-HEPES media, however, cells from the two arylsulfatase A deficient siblings showed attenuated sulfolipid catabolism. Additional clinical and laboratory studies on these individuals failed to demonstrate any features suggestive of metachromatic leukodystrophy, i.e., normal nerve conduction velocities, normal sural nerve biopsy results, and normal urinary sulfatide excretion. It is concluded that the neurologic abnormalities in the one sibling are not the result of the low enzyme activity and that both individuals represent examples of pseudo arylsulfatase A deficiency (arylsulfatase A deficiency without metachromatic leukodystrophy). These results thus call into question the ability of the high-sensitivity cerebroside sulfate loading test as carried out in MEM-HEPES media to differentiate pathologically significant defects i.e., metachromatic leukodystrophy from benign "pseudo-deficiencies."

Sulfatides in prenatal metachromatic leukodystrophy.

In one 21-week-old fetus with prenatally diagnosed metachromatic leukodystrophy, galactolipid contents were determined in the forebrain cortex, cerebellum, brainstem, spinal cord, and kidney and compared to an appropriate control. Spinal cord and kidney showed the highest sulfatide accumulation as a consequence of deficient cerebroside sulfatase activity. No increase, but a measurable amount of sulfatide, was detected in the forebrain. The prenatal neural sulfatides contained a high proportion of the hydroxy fatty acid component. The galactosyl ceramides were not reduced in the early stage of the demyelinating disease.

Retinal Pigment Epithelial Degeneration Associated with Leukocytic Arylsulfatase a Deficiency

A family exhibiting a leukocytic arylsulfatase A deficiency, probably inherited in an autosomal recessive manner, differed from patients with typical metachromatic leukodystrophy in that sulfatiduria was absent and there was readily detectable cerebroside sulfatase activity. To our knowledge, this family was unique in that there were no known members with metachromatic leukodystrophy and the only neurologic abnormality was progressive retinal pigment degeneration in the proband.

Stearoyl[1- 14C]sulfogalactosylsphingosine ([ 14C]sulfatide) as substrate for cerebroside sulfatase assay

A convenient method for large scale preparation of stearoyl[1- 14C]sulfogalactosylsphingosine has been developed. The first step consists in preparing the lysosulfatide intermediate with a good yield, which we have successfully performed. In the second step, the lysoderivative is coupled to [1- 14C]stearic acid through the acyl chloride procedure. The labeled substrate thus synthetized appears to be particularly convenient for cerebroside sulfatase determination, because of the stability of the 14C isotope, compared to the other isotopes (tritium or 35S) which have been previously used for the same purpose.

Inhibition of specific opiate binding to synaptic membrane by cerebroside sulfatase

The role of cerebroside sulfate in the specific binding of opiates to the synaptic membrane was investigated by the use of the enzyme, sulfatase A (cerebroside sulfatase, cerebroside-3-sulfate-3-sulphohydrolyase, EC 3.1.6.8). It was observed that the hydrolysis of sulfatides in the synaptic membrane by this enzyme was dependent on the presence of a heat stable protein molecule. When [ 3H]naloxone binding to the enzyme-treated membrane was determined, specific binding was attenuated. This inhibition of specific binding was dependent on the concentration of the activator molecule and could not be prevented by the addition of bovine serum albumin in the binding assay mixtures. The enzymatic pH optimum for inhibiting the specific opiate binding was between pH 4.7 and 5.0. This correlated well with the pH optimum for the sulfatase A to hydrolyze free cerebroside sulfate molecules. Scatchard analysis indicated a significant decrease in the number of binding sites with no statistically significant alteration in binding affinity of the remaining sites.

Studies in metachromatic leukodystrophy. XIII. Purification of sulfatase A from normal human liver.

Metachromatic leukodystrophy (MLD) is a genetically-determined neurolipidosis characterized by deficient activity of sulfatase A (cerebroside sulfatase) and by the resultant accumulation of cerebroside sulfate. Immunological studies using a monospecific antibody to sulfatase A have shown that MLD patients do have the enzyme protein (7). The reason why this protein has little or no activity is not yet clear. The first step in such a study is to isolate and characterize the normal sulfatase.KeywordsAryl SulfataseMetachromatic LeukodystrophyNormal Human LiverPurify Enzyme PreparationAryl Sulfatase ActiThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.